The core effector RipE1 ofRalstonia solanacearuminteracts with and cleaves Exo70B1 and is recognized by the Ptr1 immune receptor

AbstractRalstonia solanacearumdepends on numerous virulence factors, also known as effectors, to promote disease in a wide range of economically important host plants. Although some of these effectors have been characterized, none have yet been shown to target the host’s secretion machinery. Here, we used an extended library of NLR plant immune receptor integrated domains (IDs), to identify new effector targets. The screen uncovered that the core effector RipE1, of theR. solanacearumspecies complex, among other targets, associates with Arabidopsis exocyst component Exo70B1. RipE1, in accordance with its predicted cysteine protease activity, cleaves Exo70B1in vitroand also promotes Exo70B1 degradationin planta. RipE1 enzymatic activity additionally results in the activation of TN2-dependent ectopic cell death. TN2 is an atypical NLR that has been proposed to guard Exo70B1. Despite the fact that RipE1 has been previously reported to activate defense responses in model plant species, we present here aNicotianaspecies, in which RipE1 expression does not activate cell death. In addition, we discovered that RipE1 is recognized by Ptr1, aNicotiana benthamianaCC-NLR, via its cysteine protease activity. Overall, this study uncovers a new RipE1 host target and a new RipE1-activated NLR while providing evidence and novel tools to advance in-depth studies of RipE1 and homologous effectors.Author SummaryBacterial wilt disease caused byRalstonia solanacearum, poses a serious global threat for a wide range of agriculturally important plant species. This Gram-negative bacterium utilizes a collection of Type III Secretion System (T3SS) effectors to manipulate host cell defense and physiology. In this study, we searched for new subcellular plant targets of the coreR. solanacearumeffector RipE1, a cysteine protease. We discovered that RipE1 has multiple potential eukaryotic targets and further elucidated its association with the host exocyst complex. Using Artificial Intelligence (AI)-based predictions and performing bothin vitroandin plantaassays, we found that RipE1 promotes the degradation of plant exocyst component Exo70B1 through its enzymatic activity. Apart from being the first report of aR. solanacearumeffector targeting a component of the host secretion machinery, our findings also identify an NLR from a model plant species that is able to recognize RipE1 protease activity and provide evidence that can lead to the discovery of additional RipE1 targets inside the host cell.